PROJECT SUMMARY/ABSTRACT This proposal is for the purchase of a PerkinElmer Opera Phenix high-content (HCS) imaging system. There are several commercially available HCS imaging instruments available, but the Opera Phenix is a high-performance system with the necessary standard and flexible capabilities that met our criteria for the wide diversity of research studies from multiple investigators at the Broad Institute. This instrument will serve the needs of high-content screening, cell painting experiments, and detailed confocal analysis of three-dimensional structures, such as organoids and pancreatic islets. The demand for this instrument at the Broad Institute is widespread; we present the research of 11 major users and 4 minor users whose primarily NIH-funded research would be substantially positively impacted by access to this instrument. The Broad Institute currently owns one such instrument, but its usage is completely accounted for by existing partnerships (>3000 hours/year, accounting for over 9 hours/day of imaging), precluding NIH-funded users from accessing this microscope. This Opera Phenix would replace an aging (>10 years) ImageXpress Micro instrument, which lacks confocal capabilities, is significantly slower than the Opera Phenix, and has degraded in performance, resulting in many out-of-focus fields of view. The PI has substantial experience with HCS applications, and the instrument would be housed and administered by professional staff with extensive expertise using the Phenix. The Opera Phenix is known as the best-in-class HCS imager equipped with innovative, proprietary Synchrony Optics to minimize crosstalk during simultaneous acquisition for rapid collection of images with microlens-enhanced wide-view Nipkow spinning disk for true point confocality. The microscope stage is maximally flexible, and can house any ANSI/SLAS standard microplate format (6 well to 1536 well) or microscope slides for tissue studies. A unique advantage of the Opera Phenix from other HCS imagers is the water immersion objective lenses, which provide the highest capable numerical aperture (NA) available for HCS imagers. The instrument will be equipped with two 4.7 megapixel 16-bit scientific-CMOS cameras, providing a dynamic range to measure all known fluorescent probes with small and large signal-to-noise ratios, greatly enhancing the speed of image acquisition. The instrument will be equipped with a robotic automation platform for fully automated cellular and high-content screening for handling plates with stackers, incubators and liquid handling robotics. The Harmony v4.8 software manages the Opera Phenix for acquisition, analysis, and data review, and includes a new 3D image analysis module for segmentation of individual cells in 3D z-space. The software is also equipped to manage time-course experiments for kinetic measurements and tracking of individual cells, automated flat field correction, ready-made turnkey applications, fully automated cell segmentation with automatic or manual parameter tuning, and texture analysis capabilities.